Serological methods of blood tests for the diagnosis of diseases. Detection of antiviral antibodies (at) in blood serum

A simpler and more affordable approach - detection of antiviral antibodies (AT) in serum. Blood samples should be taken twice: immediately after the onset of clinical signs and after 2~3 weeks. It is extremely important to examine exactly two serum samples. The results of a single study cannot be considered conclusive due to the inability to link the appearance of AT with the present case. It is possible that these antibodies circulate after a previous infection. In such a situation, the role of the study of serum obtained during the period of convalescence can hardly be overestimated. The presence of the disease during the period of taking the first sample is indicated by at least a fourfold increase in the AT titer, which was detected during the study of the second sample.

The methods listed below do not allow differentiation of antibodies(AT) formed during illness and circulating after recovery (the duration of this period is variable for different infections). Since for adequate diagnosis it is necessary to confirm a significant increase in AT titers in two samples, the first sample is examined in the acute phase, and the second - during the recovery period (after 2-3 weeks). The results obtained are retrospective and more suitable for epidemiological surveys.

RTGA detects AT synthesized against the hemagglutinins of viruses (for example, the influenza virus). The method makes it easy to detect such antibodies (AT) in the patient's serum.

RSK- basic serodiagnosis method viral infections (among available). The reaction detects complement-fixing IgM and IgG, but does not differentiate them; to optimize the results obtained, the formulation of the reaction requires certain skills of the personnel.

REEF. If a biopsy of infected tissue is available and commercial fluorescein-labeled AT kits are available, direct immunofluorescence can confirm the diagnosis. The formulation of the reaction includes incubation of the studied tissue with AT, their subsequent removal and fluorescent microscopy of the sample.

Immunosorption methods for the detection of antiviral antibodies

Immunosorption methods(for example, ELISA and RIA) are more informative, since they detect IgM and IgG separately, which allows you to draw certain conclusions about the dynamics of the infectious process or the state of convalescence. To detect AT, known Ag is adsorbed on a solid substrate (for example, on the walls of test tubes, plastic microplates, Petri dishes) and various dilutions of the patient's serum are added. After appropriate incubation, unbound ATs are removed, enzyme-labeled antiserum against human Ig is added, the procedure for incubation and washing of unbound ATs is repeated, and any chromogenic substrate (sensitive to the action of the enzyme) is added. Since the color change is proportional to the content of specific antibodies, it is quite possible to determine their titer by spectrophotometric method. In the diagnosis of HIV infection, the immunoblotting method has found the most widespread use.

Serological examination, or, in other words, serological analysis, is the study of biological materials in the laboratory. This analysis allows you to establish the presence of pathogenic bacteria in the organism under study, or in products that undergo a control check.

Serological diagnostic methods

The study of sera or other biological objects (milk, bile, saliva, washings of the intestinal mucosa, as well as copromaterials) gives a fairly reliable idea of ​​the body's response to the introduction of an infectious agent. It should be noted that the use of serological research methods is not only of diagnostic value, but provides reliable information about the level of body protection, the state of population immunity, and the circulation of various types of rotaviruses in the region under study. The results of serological surveys can also provide information for optimizing the antigenic composition of prophylactic drugs and be used to assess the immunological efficacy of vaccines.

However, it should be noted that the need to study paired blood sera taken at intervals of 1.5-2 weeks, and the availability of express diagnostic methods reduce the diagnostic value of serological reactions.

Therefore, traditional serological tests - the complement fixation test (CFR) and the hemagglutination inhibition test (HIT) are found in present time only limited application. Nevertheless, the simplicity of these methods, the availability and cheapness of reagents make them still applicable in laboratory practice. The setting of RSK and RTGA is carried out according to generally accepted methods.

An example of the use of RSK for the study of post-vaccination immunity is the work of K. Midkhan (1989). When examining sera from 116 children aged 5 months, reliable seroconversions, according to RSK, were noted in 44% of cases, and according to the results of the neutralization test (PH) and enzyme immunoassay (ELISA) - in 83 and 96%, respectively.

Immunity Research

In order to study population immunity with the help of CSC, we examined 1246 sera of patients with OKZ aged from several months to 80 years and older. It was shown that the geometric mean titers of complement-fixing antibodies were the highest in the age groups of 2-4 and older than 60 years, which confirms our earlier data on the highest prevalence of rotavirus infection among individuals of these age groups.

Rotavirus test

RTGA is used in the study of rotavirus infection more often than RSK, and, as a rule, in combination with other laboratory tests. Thus, Andrade J. P. et al. used RTGA, immunoblot, ELISA block to study antibodies to structural proteins of rotavirus VP2, VP4, VP6 and VP7. According to the authors, in children from 2 to 4 years old, antihemagglutinating antibodies to rotaviruses were found in 70-80%.

Depending on the level of antibodies to certain proteins, according to RTGA, the authors identified 4 groups of individuals. Groups I and II (60%) included children with high levels of antibodies to VP4 and VP7 and were classified as "immune". AT III group(4%) referred persons with low level antibodies to VP7 and VP6, the so-called "partially immune". Children of group IV (36%), in whom, according to RTGA, antibodies were not detected, were designated as "non-immune" and constituted a risk group, since among them the development of severe rotavirus infection is possible. It should be noted that in this observation, the sensitivity indicators of RTGA and the ELISA block were quite comparable.

RTGA has been repeatedly used to study the structural protein VP4. Studies have shown that its hemagglutination activity is specifically inhibited by antisera, thus confirming that VP4 is the hemagglutinin of rotaviruses. Similar data were published earlier by M. Ezekiel (1995).

Thus, the presented works show that RSK and RTGA are still used in the study of rotavirus infection, however, these methods are of an auxiliary nature and should be duplicated by other tests.

Neutralization tests

The neutralization reaction is based on the ability of human or animal immune sera to neutralize the reproduction of the virus and thereby prevent the manifestations associated with this phenomenon. Depending on the biological model, these manifestations can be: the development of a disease with a specific clinic, reproduction and isolation of the virus, the production of specific antibodies, as well as the appearance of a cytopathic effect or the formation of blebs when using cell culture.

Macrobiological models are currently used mainly in veterinary medicine, the detection of viruses in humans in most cases is carried out on cell culture, both primary and transfused. PH methods are subdivided into two groups according to the principal setting scheme. In one group of methods, undiluted serum is combined with serial dilutions of the virus, in the other, serum dilutions are tested with a constant dose of virus. To set up a neutralization reaction, a pathogen that causes a pronounced cytopathic effect or is intensively reproduced in a biological model is preferable.

However, rotaviruses, unfortunately, have a mild cytopathic effect, which was convincingly demonstrated by researchers led by B. Weber (1992). A study of 121 stool samples from children with OKZ using the classical method of virus isolation on MA-104 cells under the control of CPP revealed only 4 positive cases (3.3%), while the use modern methods detection of rotaviruses (ELISA, PCR, EP in PAAG) allowed to increase this figure to 54.4%. Therefore, to increase the sensitivity of PH, additional methodological techniques are required to help visualize the reduction of the virus, which are used as: a radioactive or immunofluorescent label, plaque formation, immunoperoxidase staining, etc. It should be noted that the very principle of determining the level of virus-neutralizing antibodies is identical to the classical PH method, differing from it only in the way of resolution, that is, in the visualization of the virus-neutralizing activity of the serum. In this case, the activity of the serum is determined by its ability to suppress the manifestations of the infectious properties of the virus.

Currently, most studies use two methods to measure the amount of virus-neutralizing antibodies to rotavirus. One of them is based on counting the number of individual virus-infected cells (Plaq method) that specifically bind to fluorescent-conjugated antibodies. In another test, the level of neutralization of the virus is judged by the decrease in the production of viral antigen by the enzyme immunoassay. A comparative study of both methods showed a linear relationship between the ELISA indices and the number of plaque-forming units for each prototype strain of the rotavirus serotype: Wa, DS-1, P, VA-70. The data obtained make it easy to determine the serum dilution that provides neutralization of 60% of the infectious virus (neutralizing antibody titer). It turned out that antibody titers when testing materials by both methods are the same and both tests do not differ in reproducibility of results. Some advantage of the method using antibodies labeled with fluorescein, according to the authors, lies in the greater objectivity of the results (automated registration) and less laboriousness.

To measure the titers of virus-neutralizing antibodies, modified PH is also used by blocking ELISA with monoclonal antibodies.

• study of the intensity of the production of virus-neutralizing antibodies (VNA) during natural infection and vaccination;
• the formation of VNA to various structural proteins of rotaviruses;
• evaluating the role of VNA blood sera and secretory antibodies of saliva and intestinal mucosa in protecting against natural infection (Ward R. et al., 1990, 1992, 1993, 1995, 1997).

In conclusion, it should be emphasized that the neutralization reaction in its various modifications is still widely used both in experimental and clinical research and, with its high sensitivity and specificity, serves as a reference for all other serological methods.

Precipitation analysis

Precipitation tests are based on the interaction of serum with viral antigens using osmotic processes or under the influence of an electric field in a gel medium or other type of carrier. One of these methods is the reaction of counter immunoelectrophoresis (VIEF). The authors examined the blood sera of healthy and sick adults and children with rotavirus diarrhea, as well as specific immunoglobulin preparations for the presence of antibodies to human rotavirus by the VIEF method in 7% agarose with the addition of 4% polyethylene glycol. It turned out that antibodies to RV circulated quite widely and were found in sick adults and children in 90.2-87.7%, respectively, as well as in 78.4% of healthy children under the age of 1 year. All 32 immunoglobulin series contained antibodies to RV in titers 1:4-1:128. According to the authors, the method is suitable for studying population immunity.

radioimmunoprecipitation

Another precipitation technique is radioimmunoprecipitation. The authors used this method to study the immune response to structural and non-structural proteins in primary rotavirus infection and showed that the immune response was more pronounced to VP4 than to VP7.

When studying the serum and secretory immunological response to the use of a tetravalent reassortant vaccine in newborns For this purpose, a radioimmunoassay technique was used along with ELISA and PH. It was shown that the immune response in serum depends on the dose of the injected antigen; as for the detection of antibodies in saliva, the authors explain their appearance with the consumption of breast milk.

The radioimmunoprecipitation reaction is also used in fine research work. Thus, J. Tosser used this method to study the topology of the VP6 protein in the genome structure and suggested that VP 6 is involved in the formation of internal capsid channels.

Other researchers in 1994 also used the method of radioimmunoprecipitation to study immunological changes in rotavirus infection. The authors showed that in the acute period, mainly IgA to VP2 and VP6 are registered, while during the convalescence period, the intensity of production of secretory antibodies (IgA) not only to VP2, but also to other structural and non-structural proteins decreased. Later, similar data were obtained using the radioimmunoprecipitation method. The authors showed that, in addition to VP4 and VP7, VP2, VP6, and NSP2 are also involved in the immunological process.

Thus, immunoprecipitation has been used diagnostically to study the immune response in natural rotavirus infection.

AT last years Immunoprecipitation has been replaced by radioimmunoprecipitation, which is used to evaluate post-vaccination and post-infection immunity in RV infection and for fine-grained research and development.

hybridization methods

The immunoblot method is widely used to determine antibodies to rotaviruses. An example of the use of Western-blot in the diagnosis of rotavirus infection is the work of H. Ushijima (1989), who, using immunoblotting, characterized the specificity of antibodies to structural RV proteins in 21 children with OKZ, as well as the level of coproantibodies of IgA and IgG classes to these proteins. in uninfected children. The authors hypothesized that immunoblotting could make it possible to establish the diagnosis of the disease on a single sample of coproantibodies without examining paired blood sera. The possibility of using Western-blot to determine the level of antibodies to VP1, VP2, VP4, VP6 and VP7 was demonstrated by Pavlov I. et al. (1991). The authors examined human and animal blood sera for the presence of antibodies to rotavirus strains SA-11, DS-1, Wan Ito and concluded that Western blot can be successfully used to assess immunity in clinical practice.

It is believed that the immunoblot can be used to confirm the diagnosis of rotavirus infection without the use of paired sera for one sample of coproantibodies.

Along with RTHA and ELISA, the immunoblot was used to study herd immunity. A group of individuals who are non-immune to VP2, VP4, VP6 and VP7 have been found, which represent a risk group, primarily requiring protection by means of active and passive immunization (Andrade J. P. et al., 1996).

The use of immunoblot in the study of serological changes in the process of natural RV infection is also noted by Begue R. et al. (1998). The authors confirmed that antibodies to VP2 and VP6 are most often involved in the immune response to infection, and antibodies to VP7 and VP4 are less often involved.

Immunofluorescence analysis

An indirect immunofluorescent method using fluorochrome-labeled anti-species sera is used to titrate test sera on cells infected with rotaviruses. The reaction is based on the specific interaction of labeled antibodies and a homologous antigen, while the antigen-antibody complex is easily detected using a fluorescent microscope.

Using this method, the authors conducted a serological survey of two groups of South American Indians and found a high percentage of seropositive individuals in both groups: 67.8 and 77.4% by ELISA and 45.5 and 56.7% by IFM, respectively.

In another study on the determination of the level of antibodies to RV group C in umbilical cord blood using IFM, it was shown that 30% of women of childbearing age were detected with these antibodies, indicating a past infection.

Linked immunosorbent assay It is currently used, along with PH, in the serological diagnosis of rotavirus infection extremely widely. This method, based on the use of enzyme-labeled antibodies or antigens, is the most suitable and promising method for serological diagnosis of rotavirus infection due to its ease of implementation and economy. The method makes it possible to carry out mass seroepidemic surveys of the population, evaluate the immunological and epidemiological efficacy of vaccines, study the protective role of antibodies of various classes in various biological fluids of the human body, and also carry out serological diagnosis of rotavirus infection.

Numerous studies using ELISA were carried out by R. Azeredo (1989), which showed that the number of patients with OKZ of established rotavirus etiology was significantly lower than their level of infection according to the results of a serological examination. These data found that many clinical cases of rotavirus infection are not diagnosed by the detection of RV in faeces. Further studies on the prevalence of rotavirus infection confirmed this assumption. When conducting seroepidemiological surveys, it was found that 50-70% of the population had a high level of antibodies, which indicates the widespread circulation of RV in the human population.

Even greater opportunities are opened by ELISA in determining the dynamics of the level of antibodies belonging to different classes of immunoglobulins. So, according to the authors of the methodology in 1989, they claim that during the vaccination process, a significant increase in antibodies to RV in the blood was observed in 83-96%. Antirotavirus antibodies of the IgA and IgG classes were equally well detected by ELISA and PH by the reduction of plasma - 67.6 and 70.0%, respectively. Seroconversion of IgM class antibodies was detected in 53 and 44% of children by ELISA and RSC, respectively. Based on the analysis of the intensity of production of antibodies of different classes, the authors concluded that the most effective, simple, and fastest method for detecting seroconversions after vaccination is the method of detecting IgA antibodies using ELISA.

This conclusion was also confirmed in the work of R. Bishop (1996), which indicated that, based on the results of a survey of 68 mother-child pairs diagnosed with RV infection, it was shown that the detection of IgA antibodies using ELISA in stool materials is the most sensitive marker. both clinically expressed and asymptomatic infection. Similar results when examining children with severe RV diarrhea were obtained by J. Kolomina in 1998.

However, to study the intensity of seroconversions, it is necessary to study paired sera, which significantly lengthens the diagnostic process. At the same time, it is well known that the appearance of antibodies of the IgM class is evidence of the onset of the infectious process. According to our data, obtained during the examination of patients with rotavirus infection by ELISA and RSK, it turned out that, according to the results of ELISA, all the examined persons contained IgM antibodies to RV in their blood, while according to RSK, only 77% (R

In recent years, with the realization of the possibility of determining gene- and serotype-specific antibodies, ELISA has become a truly universal method for studying rotavirus infection, which is used:

• when studying the production of antibodies of classes IgA, M, G to individual structural and non-structural RV proteins;
• in the evaluation of immunological efficacy various kinds vaccines: attenuated, cold-adapted, DNA, reassortant;
• when studying the production of antibodies in various biological fluids of the body under conditions of natural infection and during immunization.

Thus, as follows from the presented data, the immunological aspects of rotavirus infection are studied using a wide range of laboratory methods. As a result, the choice of the optimal research method, taking into account its resolution, economic and time costs, is rather complicated and depends on the tasks facing researchers, as well as the equipment of the laboratory. And yet, from the variety of methods, in our opinion, two should be singled out - the neutralization reaction in cell culture and enzyme immunoassay - providing the study of antibodies to various classes of immunoglobulins. The possibility of express diagnostics of rotavirus infection determines the particular promise of these methods for use in wide practice.

Serology(from Latin serum - "serum", logos - "science") is a branch of immunology that studies the specifics of the interaction of serum antibodies with antigens.

The basis of diagnosis is the detection of specific antibodies that are formed as a reaction to infection of the body with a certain antigen. Depending on which antibodies are found in the blood, a conclusion is made about the nature of the infection, and the amount of these antibodies indicates the degree of activity. infectious disease.

The material taken for research as part of a serological blood test is being studied for, and others dangerous diseases- herpes, intestinal infections, rubella, toxoplasmosis, chlamydia, measles, chlamydia. In addition, this study allows you to approve the blood type, determine the specificity of proteins.

So, serological studies are carried out:

• if a preliminary diagnosis has been made and its confirmation is now required. The study is based on the addition of the appropriate antigen to the blood serum. The response allows you to make a conclusion about the presence or absence of the disease;
• if a diagnosis cannot be made. As part of the study, antibodies are added to the blood and the type of antigens is determined, which makes it possible to determine a specific disease;
• if it is needed .

Thus, a serological blood test helps to make a diagnosis or prescribe the most effective treatment- with minimal time and financial costs.

The advantages of serological studies include:

• the ability to detect pathological microorganisms in early stages infections;
• control of the development of the disease and the level of effectiveness of the therapy;
• no need for special preparation before taking the biomaterial;
• efficiency. The result will be available in two to three hours, which is very important in inpatient treatment;
• the affordability of the reagent, which allows sampling as often as needed;
• no contraindications.

How is a serological test performed?

Blood sampling is carried out from the cubital vein. Important point- blood is taken not with a syringe, but by gravity - a needle is inserted into a vein without a syringe and up to five ml of blood is collected in a test tube. The procedure is carried out in the morning.

Depending on the reactions that underlie the study, there are several types of procedures:

1. neutralization reaction. It is based on the property of immune serum antibodies to react as a neutralizing agent to microorganisms or toxins, preventing their negative effects on the body;
2. agglutination reaction. May be direct or indirect. In the first case, we are talking about the study of blood serum for the presence of antibodies (killed microbes are thrown into the material, and the reaction is evaluated - if there is a precipitate in the form of flakes, the reaction is positive), an indirect reaction is based on the introduction of erythrocytes with antigens adsorbed on them into the material ( scalloped sediment indicates a positive reaction);
3. precipitation reaction. The antigen solution is layered on the immune serum (acts as a liquid medium). Soluble antigen is used. If the antigen-antibody complex precipitates, the reaction is considered positive;
4. complement reaction. Scope — detection of infectious diseases. Complement is activated and reactions are examined;
5. reaction with labeled antigens and antibodies. It is based on the fact that tissue antigens or microbes, processed in a special way, begin to emit light under the influence of UV rays. The method is widely used not only in the diagnosis of antigens, but also for the determination of hormones, enzymes, and drugs.

It is necessary to prepare for the test: in four days the patient must refuse to take heart medications, you must also exclude alcohol in any of its manifestations, spicy and fatty foods, sweets, limit physical effort, and avoid stress.

If you do not follow these rules, the risk of false positive result. Before prescribing a second test, the doctor must find out what the patient did the day before the procedure and give recommendations on how to properly prepare for the examination.

Serological analysis: transcript

Serological blood test - an analysis that allows you to determine / confirm the type of infectious agent, helps the specialist to make a diagnosis. This is an indispensable help if the doctor cannot find drug therapy, since the causative agents of various diseases differ in different sensitivity to the action of specific drugs and antibiotics.

After the procedure for collecting the material is completed, the laboratory assistants proceed to the next stage - they decipher the indicators. So, if antibodies are not found in the patient's blood, it can be concluded that there are no infections in the body - the result of the analysis in this case is positive.

But this state of affairs is rather an exception to the rule: if there are symptoms of the disease, serological studies reveal and prove the presence of a serious pathology in the body.

First, pathogens are found in the body using an analysis, then the amount of antibodies is estimated, on the basis of which a conclusion is made about how seriously the infection is developed.

Serological testing for hepatitis, HIV, syphilis: features

Syphilis . When testing for syphilis, specialists look for proteins that are responsible for entering human body the causative agent of infection - we are talking about treponema pale. In this case, blood serum acts as a biological material.

. Viral hepatitis- serious infectious diseases, the danger of which lies in the fact that they can live in the body for quite a long time without manifesting themselves. It is possible to identify the disease in the early phase, when it is more treatable, by analyzing for markers - markers appear in the blood after the illness or the introduction of the vaccine.

Need to understand that the identification of the pathogen is possible only 1.5-2 months after infection. If the analysis is taken by a pregnant woman, a false positive result is possible.

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If you observe one or more of the symptoms listed below, you should consider taking a serological test:

• vomit;
• poor appetite or lack of it;
• causeless impotence of the body, overwork;
• yellowish complexion;
• discoloration of feces and urine.

HIV. If it showed a positive result, this does not mean that the patient is infected with AIDS. If the infection occurred recently (no more than two months ago), the presence of antibodies does not allow determining the fact of the development of the disease. A re-study is scheduled.

Serological blood test- the most important research method, the main purpose of which is the rapid detection of viruses, infections, microbes in the body.

This unique laboratory "tool" allows you to identify any disease that is a consequence of immune suppression, so do not be lazy, but regularly take an analysis in order to be able to identify the disease in time and quickly get rid of it.

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Indirect methods for diagnosing syphilis.

For the diagnosis of syphilis, many research methods are used, differing in technique and purpose. Direct methods are aimed at detecting the pathogen in the test material using microscopy (dark field microscopy, etc.) or PCR.

In addition to the methods of direct detection of pale treponema (T. pallidum), when examining for syphilis, indirect ( serological) research methods that detect antibodies to the causative agent of syphilis in blood serum and cerebrospinal fluid. Indirect methods for diagnosing syphilis include serological tests of ELISA, RPHA, CSR, RIF and RIF-abs, RIBT, PCR, express method, immunoblot, and others.

Serological methods for diagnosing infectious diseases.

Serological diagnostic criteria based on specific antigen-antibody reactions. Antigens (components of cell walls, flagella, capsules, DNA and toxins) of those microorganisms that need to be determined react with antibodies contained in sera. Binding occurs between antigens and their corresponding antibodies, which is the basis of serological diagnostic methods. Serological tests are used to detect an unknown antigen (source of which is a bacterium, virus, toxin, etc.) using a known antibody or to detect an antibody in serum using a known antigen.

Serological reactions are classified depending on the state of the antigen and the characteristics of the environment in which the antigen and antibody interact, as well as according to the method of carrying out.

To conduct serological reactions, many laboratory methods are used, such as agglutination, precipitation, complement fixation, immunofluorescence, enzyme immunoassay and radioimmunoassay, and others. These reactions allow efficient preliminary identification of microorganisms.

The sera required for setting up serological reactions are obtained experimentally, in particular, they are produced by the institutes of vaccines and sera, and are offered as part of commercial diagnostic kits.

Serological methods are an important tool in the diagnosis and treatment of infectious diseases in humans and animals, since they can not only identify the causative agent of the disease, but also detect specific antibodies to the corresponding pathogens in the blood of patients and recovered patients. Serological methods are currently the most effective methods diagnostics when it is impossible or difficult to isolate the pathogen, and relatively rarely give false positive and false negative results.

Using serological methods to diagnose syphilis

The basis of serological laboratory methods for diagnosing syphilis is the ability of the body to give an immune response to the appearance of the pathogen. With the help of serological reactions in the serum (plasma) of the blood (or cerebrospinal fluid), traces of the immune response to infection are detected, namely antibodies to the antigens of pale treponema, or the antigens themselves.

In order to establish the specific nature of the existing clinical manifestations(i.e. that they are a consequence of syphilis) and, in the future, to control the dynamics of the pathological process, it was proposed a large number of serological research methods. These same methods are also widely used for population screening.

In accordance with current national guidelines and clinical guidelines, in many countries of the world, advanced serological methods are used in population surveys to identify patients with syphilis and in establishing a clinical diagnosis. They are used both in the presence of clinical signs of the disease in patients, and in latent periods.

Setting serological reactions is one of the most reliable methods for diagnosing syphilis. Standardized serological tests with a regulated method of setting are called serological tests for syphilis. Considering that almost all serological reactions to syphilis under certain conditions can be false positive or false negative, they should be put in a complex and, if necessary, in dynamics.

Serological tests, which are used to determine antibodies in the patient's blood serum, differ from each other in sensitivity, specificity, complexity of setting and cost. In addition to classical methods, immunological tests and technologies are used for effective syphilis serodiagnosis, which have received a new impetus to development in the 21st century.

Antibodies are detected by various immunochemical (serological) methods, including sedimentary reactions, enzyme immunoassay, immunochemiluminescence, immunochromatographic assays, linear immunoblotting, flow fluorometry, immunochip technology, and others.

Serodiagnosis (a study using serological methods) is used for:

• confirmation of the clinical diagnosis of syphilis,
• diagnosis of latent syphilis,
• monitoring the effectiveness of treatment, as one of the criteria for the cure of patients with syphilis,
• prevention of syphilis (screening examination of certain groups of the population in order to identify pathology).

The most important properties required by serological tests - sensitivity, specificity, reproducibility

The decisive criterion for choosing a method laboratory diagnostics is its effectiveness - sensitivity, specificity and reproducibility.

Sensitivity is the proportion of positive results in patients. The sensitivity of the method is determined by the percentage of positive results of the study of samples that are known to contain specific markers of the pathogen (eg, pathogen antigens or antibodies to them).

Specificity- proportion of negative test results in healthy patients. The specificity of the method is determined by the percentage of negative results of the study of samples that obviously do not contain specific markers of the pathogen. Thus, the higher the sensitivity and specificity, the more reliable and reliable the research method.

To important diagnostic criteria also applies reproducibility results from repeated examinations of the same samples. It should be noted that, despite significant advances in high technology, there are no methods that provide 100% (absolute) sensitivity and specificity in all bioassays studied.

Currently, test systems, kits, and ingredients that have less than 95% sensitivity and specificity when setting up a reaction are officially registered in Russia. Thus, there is always the possibility of obtaining an inadequate result.

Classification of tests according to the type of antigen used. Treponemal and non-treponemal tests for syphilis

In modern venereology, more than a dozen variants of serological reactions to syphilis are used for diagnostic purposes, which can be divided into different categories according to different criteria. The classification is carried out according to the research methodology, scope, speed, low cost, requirements for laboratory equipment, etc.

Treponemal reactions are theoretically more specific, but they also give false positive results. Moreover, they do not allow to differentiate between untreated and cured syphilis. The results of treponemal reactions will be positive in both cases. Non-treponemal reactions distinguish between untreated or recent and cured infections.

During the examination, it is recommended to carry out both treponemal and non-treponemal reactions. To establish and confirm the diagnosis of syphilis, positive results are required for both types of tests - treponemal and non-treponemal. Therefore, non-treponemal tests are used in combination with treponemal tests, and are carried out before, during and after the end of treatment at certain time intervals.

1. Non-treponemal tests

The most famous of the non-treponemal tests with a visual interpretation of the results are

• RW - Wassermann reaction with lipid antigens (complement fixation reaction with cardiolipin antigen, RSKk)
• Kann reaction (not currently used),
• cytocholic reaction of Zaks-Vitebsky (not currently used),
• microreaction of precipitation with plasma or inactivated serum (MRP or RMP),
• RPR (Rapid Plasma reagin test),
• TRUST (Toluidin Red Unheated Serum Test).

Among non-treponemal tests, there are 2 tests with microscopic reading of reaction results:

1. VDRL - (Venereal Disease Research Laboratory);

2. USR - test for the determination of active plasma reagins (Unheated Serum Reagins).

Reactivity in nontreponemal tests usually indicates tissue damage and is not always specific for syphilis. Ease of implementation and low cost allows them to be used as screening reactions in establishing a preliminary diagnosis of syphilis.

2. Treponemal tests

Treponemal tests use specific treponemal antigens. These tests are required to confirm the diagnosis (RPHA, RIT, RIF and ELISA). They are more complex and expensive than Group 1 tests, but also more specific and sensitive. The detection of antibodies in the cerebrospinal fluid is also performed using treponemal tests.

Traditional treponemal tests confirming the diagnosis of syphilis require expensive laboratory equipment and experienced personnel, so they are rarely performed outside of specialized laboratories. However, they can now be replaced by simple and rapid treponemal tests that can be carried out in the field and use whole blood. Carrying out these reactions does not require long-term training, special conditions for the storage of reagents and equipment.

The comparative cheapness, convenience and practicality of treponemal express reactions draw attention to them not only as methods of confirming the diagnosis. These reactions can be used to screen for syphilis as part of primary care. medical care(they can be done in the field, in the same health care facility) or in areas where laboratories are not available. However, since antibodies to T. pallidum are determined over a number of years, regardless of whether the patient was treated or not, treponemal rapid reactions cannot be used to assess the effectiveness of treatment and differential diagnosis untreated and cured syphilis.

Classification of serological methods for diagnosing syphilis according to the type of antibodies detected

In modern dermatovenereology, various serological reactions to syphilis are used for diagnosis. Some of the methods that were relevant ten years ago are no longer used due to complexity or lack of specificity. Depending on the detected antibodies, the methods of serological diagnosis of syphilis are divided into three groups:

I. Lipid (reagin) reactions - antibodies to lipid antigens (reagins) are determined:

1) flocculation: microreaction on glass with lipid antigens - an express diagnostic method (precipitation microreactions - MRP), VDRL, CMF (cardiolipin microflocculation test), RPR, etc .;

2) complement fixation reaction (RSK) with lipid antigens: Wasserman reaction (RV), qualitative and quantitative method of setting, thermostatic and in the cold (Colmer reaction);

3) sedimentary reactions that are not currently used: the Cahn precipitation reaction, the cytocholic Sachs-Vitebsky reaction, etc.;

II. Group treponemal reactions - antibodies to group treponemal antigens (which are part of the microbial cell of both pathogenic and saprophytic treponemas) are determined:

1) CSC with Reiter's protein antigen;

2) immunofluorescence reaction (RIF);

3) immune adhesion reaction (RIP).

III. Species-specific protein treponemal reactions - antibodies to specific species antigens of Treponema pallidum are determined:

1) reaction of immobilization of pale treponemas (RIT);

2) RIF-abs immunofluorescence reaction and its variants (IgM-FTA-ABS, 19S-IgM-FTA-ABS, etc.);

3) reaction of indirect hemagglutination of pale treponemas (TPHA) and its modification TPPA.

4) enzyme immunoassay (ELISA);

5) immunoblotting.

Practical use of serological tests for syphilis

Different serological tests are used for different practical purposes.

Abroad, in mass surveys of the population and, if necessary, emergency detection of syphilis, non-treponemal screening reactions (VDRL, RPR, etc.) are used. Diagnosis requires confirmation by treponemal tests FTA-ABS, TPHA, or TPPA. Currently, it is recommended to use ELISA as a replacement for VDRL in screening tests. This is due to the fact that the ELISA test is distinguished by sensitivity, specificity, the ability to automate research, as well as the development of diagnostic test kits. In addition, a reverse scheme of the order of examination for syphilis is substantiated, when treponemal tests are used first.

In order to monitor the effectiveness of therapy and to assess the activity of the infection, quantitative VDRL is recommended. An ELISA test for antitreponemal IgM antibodies is used as a confirmatory/additional test.

In domestic practice, a complex of serological reactions (CSR) is used, including a microprecipitation reaction (RMP) with cardiolipin antigen and CSC with cardiolipin and treponemal antigens. Recently, it is recommended in the CSR to replace RSK with ELISA or RPHA. RIF (and its modifications - RIF-Abs and others), RIBT are also used.

RIBT is used as an examination reaction in cases of divergence of treponemal reactions.

Approaches to laboratory serological diagnosis of syphilis in the Russian Federation

Introduction into practice of unified methods of clinical laboratory research in the USSR and Russian Federation allowed for more progressive diagnostic methods, to streamline the work of clinical diagnostic laboratories, increase labor productivity, reduce duplication of laboratory tests and became the basis for the development of rational forms of ready-made reagent kits.

In 1985, in the USSR, in order to improve the diagnosis of syphilis, the non-treponemal CSC reaction with a nonspecific (Wassermann) antigen and sedimentary reactions (cytocholic and Cana) were excluded from the diagnostic complex as less sensitive and not providing additional information.

Instead, as part of the complex of serological tests for syphilis (CSR), the use of the complement fixation reaction with treponemal and cardiolipin antigens (RSKt) and the microprecipitation reaction with cardiolipin antigen (RMP) was provided. The higher sensitivity and information content of this complex of reactions ensured the detection of not only reagins, but also antitreponemal antibodies.

1985

1. RMP with cardiolipin antigen with blood plasma and inactivated blood serum. Selection test in case of isolated application.

2. CSC with treponemal and cardiolipid antigens; qualitative and quantitative methods of setting, thermostatic and in the cold;

3. Treponema pallidum immobilization reaction (RIT); test-tube and melange methods of setting;

4. Immunofluorescence reaction (RIF) in the following modifications: RIF with absorption (RIF-abs) with blood serum and capillary blood, RIF-200, RIF with whole cerebrospinal fluid (RIF-c); qualitative and quantitative methods of setting. Diagnosis of latent and late forms of syphilis, recognition of decision makers (false-positive results)

5. Complex of classical serological reactions (CSR): RSC (Wasserman reaction) with cardiolipin and treponemal antigens + RMP. Preventive examination of the population for syphilis, diagnosis of all forms of syphilis, monitoring the effectiveness of treatment, examination of persons who are contacts for syphilis.

In 2001, the Ministry of Health of the Russian Federation approved a new regulatory document regulating the procedure for conducting diagnostic tests - order of the Ministry of Health of the Russian Federation No. 87 dated March 26, 2001 "On improving the serological diagnosis of syphilis".

In order to improve the laboratory diagnosis of syphilis, improve the quality of work and prevent further dissemination the incidence of syphilis, it is recommended to replace RSK in the complex of seroreactions (CSR) in the diagnosis of syphilis by ELISA and TPHA as screening and confirmatory tests, because. these test systems are highly sensitive, specific and reproducible.

Order No. 87 of March 26, 2001 “On improving the serological diagnosis of syphilis” provides for the use of the following methods for sero- and cerebrospinal fluid diagnosis of syphilis in Russia:

1. RMP and foreign analogues (VDRL, RPR and similar microreactions) as screening tests when examining the population for syphilis. RMP is performed with plasma or inactivated blood serum.
2. Enzyme immunoassay (ELISA). Antigen from cultured or pathogenic treponema pallidum. Diagnostic reactions, including for liquor diagnostics. Due to the ease of setting and the availability of commercial test systems, they can be used as screening tests.
3. The reaction of passive hemagglutination (RPHA). Antigen from cultured or pathogenic treponema pallidum. Selection and diagnostic reactions.
4. Qualitative and quantitative variants of RIF (RIF-abs, RIF-c, RIF with capillary blood from a finger). Antigen - pathogenic pale treponema of the Nichols strain.
5. A complex of serological reactions (CSR) for syphilis, consisting of a complement fixation reaction (CFR) with treponemal and cardiolipin antigens, and RMP. It is possible to replace the complement fixation reaction with ELISA or RPHA also in combination with RMP. CSR refers to diagnostic tests.
6. Pale treponema immobilization reaction (RIBT), in which pathogenic treponema pallidum of the Nichols strain is used as an antigen. RIBTs are diagnostic confirmatory tests.

Thus, the sequence of examination of patients for syphilis in healthcare facilities is recommended to be planned as follows:

1. During the initial examination, a selection (screening) reaction of microprecipitation (RMP) or its modification (RPR, TRUST, VDRL) is performed in quantitative and qualitative versions and, in case of a positive result, any specific confirmatory treponemal test (RPHA, ELISA, CSR, RIF , RIT);

2. After the end of therapy, RMP or its modification is placed, and the dynamics of the infectious process and the effectiveness of therapy are judged by the decrease in titer. A confirmation of the effectiveness of the therapy is a decrease in titer by 4 or more times within 1 year.

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What are serological tests, why are they carried out, what are the main methods of serological diagnostics?

Serological studies are methods for studying antigens or antibodies in the biological material of patients, based on certain immune reactions. Detection of antibodies to the causative agent of infection or antigens in the biological material makes it possible to establish the cause of the disease.

do not have 100% specificity and sensitivity in the diagnosis of infectious diseases. Therefore, their results should be evaluated taking into account clinical picture diseases. This necessitates the use of several tests to diagnose infection, as well as the use of methods western blot confirming the results of screening methods.

“Immunoblotting (Western blot, Western-blot) is actually the final verification (confirmatory) method in the chain of immunological studies that allow you to make a final laboratory conclusion. This test is especially important for confirming or refuting an infectious disease (for example, whooping cough, borreliosis, etc.). Western blot is usually needed after detection positive antibodies class IgG infectious process, tk. it is in this combination that a more correct laboratory interpretation of the results and further tactics of treating the patient occur. For the Western blot, special nitrocellulose strips are used, onto which proteins are transferred by horizontal and then vertical immunophoresis in order of increasing molecular weight. The patient's serum antibodies interact with proteins in certain areas of the strip, and then the course of the reaction is similar to enzyme-linked immunosorbent assay (ELISA)”,- explains Candidate of Medical Sciences, Medical Director of the Laboratory of SYNEVO Ukraine, Oksana Vladislavovna Nebyltsova.

Biological material used for serological testing

• Serum
• Saliva
• Fecal matter

What conditions are serologic tests used to diagnose?

The purpose of serological studies is to establish the effectiveness of the treatment after the patient's recovery, as well as to detect a relapse of the disease. In addition, serological methods can detect diseases such as:

• Amoebiasis
• Giardiasis
• Opisthorchiasis
• Trichinosis
• Toxocariasis
• Cysticercosis
• Echinococcosis

Main methods used in serological diagnosis

Immunofluorescence reaction (RIF, Koons method)

Varieties of the method:

Direct: microbes or tissue antigens or microbes are treated with special sera with antibodies, as well as labeled fluorochromes that glow under UV light. Bacteria thus glow in the form of a green border around the cell periphery. observed under a fluorescent microscope.

Indirect: swabs are treated with antimicrobial rabbit serum antibodies. Then the antibodies that have not bound to the antigens of the microbes are washed away. Thus, the antibodies remaining on the microbes are detected. As a result, a complex of microbe + anti-rabbit antibodies + antimicrobial rabbit antibodies labeled with fluorochrome is formed. This complex is observed under a fluorescent microscope.

To evaluate the results, there is a four-point scale, which is characterized by the intensity of the surface yellow-green glow of antigen cells:

Very weak cell glow

Weak glow of the cell periphery

+++/++++ bright cell glow

The reaction titer is considered to be a dilution of serum with a reaction result of +++ or ++++.

The reaction of indirect hemagglutination (RNG)

The reaction of passive or indirect hemagglutination is used when diagnosis of infections caused by protozoa, bacteria and rickettsiae.

The technique consists of several stages. First, the erythrocytes are processed, “washed” with an isotonic sodium chloride solution, then, if necessary, with a 1: 20,000 tannin solution, then they are sensitized with soluble antigens. After treatment with a buffered isotonic sodium chloride solution, the antigen is ready for use. The serum is diluted in test tubes with isotonic sodium chloride solution, after which an erythrocyte diagnosticum is added to each diluted serum.

The results are evaluated by the nature of the erythrocyte sediment:

Weak intensity

Average intensity

intense reaction

A sharply intense reaction

A positive result is a reaction with an intense reaction +++ or a sharply intense reaction ++++, in which erythrocytes cover the entire bottom of the test tube.

Enzyme immunoassay (ELISA)

The method has high specificity and sensitivity, more than 90%. The main advantage is the possibility detection of infection and tracking the dynamics of the process, which indicates the level of antibodies.

The assay is used to diagnose a wide range of infections, including:

• HIV infection
• Viral hepatitis
• Cytomegalovirus infection
• herpes infection
• Toxoplasma infection

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The antigen or antibody is fixed on solid plates, then, using an enzyme label, antigen-antibody complexes are detected.

ELISA has a number of advantages over others serological methods. The reaction is the most sensitive, and universal reagents are used in the tests. The analysis is fast, the ability to study immunoglobulins of various classes. Currently, ELISA is one of the main methods of laboratory diagnostics.

Evaluation of the results of ELISA is carried out automatically using special devices. In some cases, a visual account of the results of the reaction is also allowed. The reliability of serological diagnosis depends on the organization of laboratory control, consisting of several procedures designed to assess the quality of the results.